DESCRIPTION: (Adapted from the applicant's abstract.) The proposed studies are directed to an examination of the mechanism of endothelial cell retraction. The hypothesis directing this work is that endothelial cell (EC) retraction requires two events for the formation of retractive gaps: a dissolution or gel-sol transition in the cortical cytoplasm and the activation of the EC contractile apparatus. Ethchlorvynol (ECV), a chemical mediator which causes Adult Respiratory Distress Syndrome (ARDS), and histamine and thrombin, two important mediators of inflammation that increase vascular permeability will be used to test their hypothesis. A tissue culture model utilizing human vein and bovine pulmonary artery endothelial cells will be used to simulate the retraction that occurs in vivo in ARDS. The studies have been designed to determine if retraction and gap formation are associated with EC myosin light chain phosphorylation. The relationships between MLC phosphorylation, cytosolic Ca2 positive, cellular deformability and monolayer permeability will be determined. In addition, the enzymes (MLCK or PKC or both) responsible for phosphorylating MLC in agonist stimulated monolayers will be identified. Elevation of cyclic adenosine monophosphate and the use of synthetic peptide inhibitors to myosin light chain kinase will be employed to inhibit EC retraction, MLC phosphorylation and cellular deformability. The role of gelsolin, a Ca2 positive activated actin severing protein, in the changes in the structure of the actin network will be investigated. The group will correlate the EGTA resistant 1:1 actin/gelsolin complexes and actin severing activity from agonist stimulated monolayers. The time course for formation of actin/gelsolin complexes and actin severing activity will be compared with Ca2 positive mobilization, cellular deformability and EC retraction. The permeabilized EC preparation will be used to test the effects of gelsolin on EC retraction. Varying concentrations of gelsolin will be added to these preparations and the effect on the actin network and retraction determined. Confocal fluorescence microscopy will be used to define the three-dimensional characteristics of the actin network with particular attention to the peripheral rim of actin. The structure of the cytoskeleton in resting and stimulated EC will be defined with respect to the disposition of actin, myosin, MLCK and gelsolin.